Team:MIT/Tooth binding assay protocol

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Revision as of 15:40, 27 June 2008

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Tooth binding assay

Wash 50 mg HA beads 3 times with 1mM phosphate buffer (PB) and equilibrate in PB for 2 hours (or overnight)

HA beads added to 500 microL saliva and stir suspension for 1h at 37 C

       Aspirate saliva and wash s. mutans with 10x PBS
   
       Dilute bacteria with 1x PBS to get the final concentration of 1-2 *10^7 CFU (.075 to .16 OD--use .10)

Add 1mL S. Mutans suspension to HA beads, incubate at 37 C at 20 rpm in microfuge tubes

Extract 100 μL supernatant after 5 minutes and spot onto plates (10g agar, 15g THB, 500 mL H20)

Repeat above after 1h, 2h

Count colonies (approximately CFUs) on the three plates and calculate the amount attached to HA beads through the relation “CFU supernatant time 0 – CFU supernatant time 1h, 2h = CFU on HA beads"

PBS—naCL 8.0 g L-1, KCl 2.0 g L-1, Na2HPO4, 2H20 2.0 g L-1, KH2PO4 2.0 g L-1; pH 7.2

OD-CFU conversion factor: optical density at 600 nm of 0.75 to 0.8: 1 × 108 CFU/ml)

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@@ Line 21: / Line 21: @@
          Aspirate saliva and wash s. mutans with 10x PBS
-         Dilute bacteria with 1x PBS to get the final concentration of 1-2 *10^7 CFU (.075 to .16 OD)
+         Dilute bacteria with 1x PBS to get the final concentration of 1-2 *10^7 CFU (.075 to .16 OD--use .10)
 	Add 1mL S. Mutans suspension to HA beads, incubate at 37 C at 20 rpm in microfuge tubes